Methods for treating skin pigmentation

ABSTRACT

This invention relates to methods and compositions for bringing about changes in skin pigmentation. More particularly, this invention relates to compounds which affect melanogenesis and can be used as depigmenting agents or as agents for darkening skin utilizing the PAR-2 pathway.

[0001] This is a continuation-in-part application of U.S. patentapplication Ser. No. 09/110,409 (Attorney Docket No. JBP 430), which ishereby incorporated herein by reference.

BACKGROUND

[0002] 1. Field of the Invention

[0003] This invention is related to methods and compositions forbringing about skin pigmentation and/or for causing skin depigmentation.More particularly, this invention relates to compounds which affectmelanogenesis and can be used as depigmenting agents or as agents fordarkening skin.

[0004] 2. Background of the Invention

[0005] Skin coloring has been of concern to human beings for many years.In particular, the ability to remove hyperpigmentation, such as found inage spots, freckles or aging skin generally, is of interest toindividuals desiring a uniform complexion. In certain areas of theworld, general body whitening is desirable. There are alsohypopigmentation and hyperpigmentation disorders that are desirable totreat. Likewise, the ability to generate a tanned appearance withoutincurring photodamage due to solar radiation is important to manyindividuals. There have been many methods proposed to accomplishdepigmentation, as well as to accomplish darkening of the skin. Forexample, kojic acid, hydxocione, retinoids and other chemical compoundshave been used for depigmentation. Dihydroxyacetone and like chemicalcompounds have been utilized for their ability to “tan” the skin withoutexposure to the sun.

[0006] Many of these previous solutions have not been found acceptable.There is often a distinct line of demarcation between the areas of skinto which such previous compositions have been applied. Therefore,precise application of all these compounds is necessary in order toachieve the desired result. Many of these compounds have been found tobe quite irritating to the skin and therefore undesirable for use.

[0007] The understanding of the chemical and enzymatic basis ofmelanogenesis is heavily documented. Melanocytes migrate from theembryonal neural crest into the skin to produce secretory granules,melanosomes, which produce melanin. Melanogenesis occurs within themelanosome, and the melanin is later distributed to keratinocytes viathe melanocyte dendrites. The key enzyme in melanogenesis is tyrosinase,which initiates a cascade of reactions which convert tyrosine to thebiopolymer melanin. Two tyrosinase-related proteins (TRP's) are known,TRP-1 and TRP-2. These proteins share with tyrosinase about 40% homologyand have catalytic activities as well as regulatory roles inmelanogenesis. TRP-1 is the most abundant glycoprotein in melanocytes.

[0008] In spite of the fact that the chemical and enzymatic basis ofmelanogenesis is well-documented, its regulation at the cellular levelis only partially understood. Tyrosinase and the TRP's share structuraland biological properties with the lysosomal-associated membrane protein(LAMP) gene family, therefore their targeting to the melanosomalmembrane might induce their activation. Aphosphorylation/dephosphorylation reaction at the cytoplasmic tails ofthese proteins could be involved in the regulation of melanogenesis. Thebeta isoform of the Protein Kinase C (PKC) family has been shown toregulate human melonogenesis through tyrosinase activation. Geneexpression of tyrosinase, TRP-1 and TRP-2 is coordinated. All threeenyzmes are expressed in human epidermis. In melanocytes co-culturedwith keratinocytes, these transcripts are expressed at a ratio of45:45:10, respectively. In melanocytes cultured alone, only TRP-1transcripts are present, indicating that a keratinocyte-derived signalis involved in the coordinate expression of these genes. The regulationof keratinocyte-melanocyte interactions and the mechanism of melanosometransfer into keratinocytes are not yet understood.

[0009] The Protease-activated receptor-2 (AR-2) is a seven transmembraneG-protein-coupled receptor, that is related to, but distinct from thethrombin receptors (TR, also named PAR-1, and PAR-3) in its sequence.Both receptors are activated proteolytically byan arginine-serinecleavage at the extracellular domain. The newly created N-termini thenactivate these receptors as tethered ligands. Both receptors could beactivated by trypsin, but only the TRs are activated by thrombin. OnlyPAR-2 is activated by mast cell tryptase. Both receptors could also beactivated by the peptides that correspond to their new N-termini,independent of receptor cleavage. SLIGRL, the mouse PAR-2 activatingpeptide, is equipotent in the activation of the human receptor. Whilethe function of the TR is well documented, the biology of the PAR-2 hasnot yet been fully identified. A role for PAR-2 activation in theinhibition of keratinocyte growth and differentiation has been recentlydescribed (Derian et al., “Differential Regulation of Human KeratinocyteGrowth and Differentiation by a Novel Family of Protease-activateReceptors”, Cell Growth L & I Differentiation, Vol. 8, pp. 743-749, July1997).

SUMMARY OF THE INVENTION

[0010] In accordance with this invention, we have found a method foraffecting changes in mammalian skin pigmentation comprising topicallyapplying to the skin of a mammal a compound which affects the PAR-2pathway. The compositions of this invention may contain one or morecompounds that act as trypsin, as tryptase, as serine protease or asPAR-2 agonists, for increase in pigmentation. Alternatively, they maycontain one or more compounds that act as serine protease inhibitors,trypsin inhibitors, thrombin inhibitors, tryptase inhibitors, as PAR-2pathway inhibitors or as a PAR-2 antagonist for decrease inpigmentation, or “depigmentation”.

[0011] As used herein, “mammal” means any member “of the highervertebrate animals comprising the class “Mammalia”, as defined inWebster's Medical Desk Dictionary 407 (1986), and includes but is notlimited to humans. As used herein, “receptor” shall include bothintracellullar and extracellular receptors and shall mean thosemolecules capable of receiving and transducing a signal. The term PAR-2refers to the protease-activated receptor-2 or a related proteaseactivated receptor. The Protease-activated receptor-2 (hereinafter,“PAR-2”) is a serine-protease activated receptor that is expressed innumerous tissues, including keratinocytes and fibroblasts. The thrombinreceptor (also named PAR-1, hereinafter, “TR”) is a serine-proteaseactivated receptor that is expressed in numerous tissues, includingkeratinocytes. The biological roles of PAR-2 and TR in skin are notentirely known. However, we have found that interactions betweenkeratinocytes and melanocytes, via the PAR-2 pathway, affectmelanogenesis. We have found that thrombin inhibitors, and/or tryptaseinhibitors, and/or trypsin inhibitors and PAR-2 antagonists can be usedas depigmenting agents without irritation of the skin. PAR-2 agonistsand serine proteases such as trypsin and tryptase can be used asdarkening agents. Furthermore, PAR-2 could be useful as a target forwhitening and darkening agents.

[0012] We have further discovered that BBI, a Bowman-Birk typeinhibitor, may also be used as an active depigmenting agent.Soybean-derived extracts and mixtures that were suggested in U.S. patentapplication Ser. No. 09/110,409 as depigmenting agents contain both STIand BBI. We have now found that BBI alone is effective to depigmentskin. BBI may be used in all the formulations and compositions set forthin the parent application in the same range of concentration as STI.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 shows epidermal equivalents containing melanocytes of anAfrican-American donor. Treatment with BBI reduces pigment deposition inthese equivalents, as demonstrated by top view of the equivalents, withno staining.

[0014]FIG. 2 shows epidermal equivalents containing melanocytes of anAfrican-American donor. Treatment with BBI reduces pigment deposition inthese equivalents, as demonstrated by Fontana-Mason staining ofhistological sections of these equivalents.

[0015]FIG. 3 shows epidermal equivalents containing melanocytes of anAfrican-American donor. Treatment with increasing concentrations of BBIreduces pigment deposition in these equivalents in a dose-dependentfashion, as demonstrated by Fontana-Mason staining of histologicalsections of these equivalents.

[0016]FIG. 4 is a graph quantifying the percent of inhibition of pigmentdeposition following BBI treatment.

[0017]FIG. 5 shows F&M stained histological sections from swine skintreated with BBI and STI. Melanin deposition in the swine skin isdramatically reduced following BBI or STI treatment.

[0018]FIG. 6 is a graph of computerized image analysis of pigmentdeposition in skin sections such as those demonstrated in FIG. 5. Thegraph quantifies the percent of inhibition of pigment deposition in theswine skin following BBI or STI treatment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] We have discovered that trypsin, tryptase and PAR-2 agonists canbe used to increase pigmentation and that trypsin inhibitors, and/ortryptase inhibitors, and/or thrombin inhibitors and PAR-2 antagonistsact to decrease pigmentation in mammalian skin. In our opinion, some ofthe compounds described in U.S. Pat. No. 5,523,308, which is herebyincorporated herein by reference, and behave as thrombin and/or trypsinand/or tryptase inhibitors, will be useful in methods of this invention.Some of these compounds are also described in Cosanzo, et al., “PotentThrombin Inhibitors That Probe the S₁′ Subsite: Tripeptide TransitionState Analogues Based on a Heterocycle-Activated Carbonyl Group”, J.Med. Chem., 1996, Vol. 39, pp. 3039-3043 and have the followingstructural formula:

[0020] wherein:

[0021] A is selected from the group consisting of C₁₋₈alkyl,carboxyC₁₋₄alkyl, C₁₋₄alkoxycarbonyC₁₋₄alkyl, phenylC₁₋₄alkyl,substituted phenylC₁₋₄alkyl (where the phenyl substituents areindependently selected from one or more of, C₁₋₄ alkyl,perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido, nitro amino,C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy or C₁₋₄ alkoxycarbonyl),fornyl, C₁₋₄alkoxycarbonyl, C₁₋₂alkylcarbonyl, phenylC₁₋₄alkoxycarbonyl,C3-7cycloakylcarbonyl, phenylcarbonyl, substituted phenylcarbonyl (wherethe phenyl substituents are independently selected from one or more of,C₁₋₄alkyl, perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido, nitro,amino, C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy or C₁₋₄alkoxycarbonyl),C₁₋₄alkylsulfonyl, C₁₋₄alkoxysulfonyl, perfluoroC₁₋₄alkyl-sulfonyl,phenylsulfonyl, substituted phenylsulfonyl (where the phenylsubstituents are independently selected from one or more of, C₁₋₄alkyl,perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido, nitro, amino,C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy or C₁₋₄alkoxycarbonyl),10-camphorsulfonyl, phenylC₁₋₄alkylsulfonyl, substitutedphenyC₁₋₄alkylsulfonyl, C₁₋₄alkylsulfonyl, perfluroC₁₋₄alkylsulfonyl,phenylsulfinyl, substituted phenylsulfinyl (where the phenylsubstituents are independently selected from one or more of, C₁₋₄alkyl,perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido, nitro, amino,C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy or C₁₋₄alkoxycarbonyl),phenylC₁₋₄alkylsulfinyl, substituted phenylC₁₋₄alkylsulfinyl,1-naphthylsulfonyl, 2-naphthylsulfonyl or substituted naphthylsulfonyl(where the naphthyl substituents are independently selected from one ormore of, C₁₋₄alkyl,perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido,nitro, amino, carboxy or C₁₋₄alkoxy-carbonyl), 1-naphthylsulfinyl,2-naphthylsulfinyl or substituted naphthylsulfinyl (where the naphthylsubstituents are independently selected from one or more of, C₁₋₄alkyl,perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido, nitro, amino,C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy or C₁₋₄alkoxycarbonyl);

[0022] a D or L amino acid which is coupled as its carboxy terminus tothe nitrogen depicted in formula I and is selected from the groupconsisting of alanine, asparagine, 2-azetidinecarboxylic acid, glycine,N-C₁₋₈alkyglycine, proline, 1-amino-1-cycloC₃₋₈alkycarboxylic acid,thiazzolidine-4-carboxylic acid, 5,5-dimethylthiazolidine-4-carboxylicacid, oxadolidine-4-carboxylic acid, pipecolinic acid, valine,methionine, cysteine, serine, threonine, norleucine, leucine,tert-leucine, isoleucine, phenylalanine, 1-naphthalanine,2-naphthalamine, 2-thienylalanine, 3-thienylalanine,[1,2,3,4]-tetrahydroisoquinoline-1-carboxylic acid and1,2,3,4,]-tetrahydroisoquinoline-2-caroboxylic acid

[0023] where the amino terminus of said amino acid is connected to amember selected form the group consisting ofC₁₋₄alkyl,tetrazol-5alkyl-C₁₋₂alkyl, carboxytC₁₋₄alkyl,C₁₋₄alkoxycarbonylC14phenylC₁₋₄alkyl, substituted phenyl C₁₋₄, alkyl(where the phenyl substituents are independently selected from one ormore of, C₁₋₄alkyl, perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo,amido, nitro, amino, C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy orC₁₋₄alkoxycarbonyl), 1,1-diphenylC₁₋₄alkyl, 3-phenyl-2-hydroxypropionyl,2,2-diphenyl-1-hydroxyethylcarbonyl,[1,2,3,4]-tetrahydroisoquinoline-1-carbonyl,[1,2,3,4]-tetrahydroisoquinoline-3,carbonyl,1-methylamino-1-cyclohexanecarbonyl, 1-hydroxy-1-cyclohexanecarbonyl,1-hydroxy-1-pheny-lacetyl, 1-cyclohexyl-1-hydroxyacetyl,3-phenyl-2-hydroxypropionyl, 3,3-diphenyl-2-hydroxypropionyl,3-cyclohexyl-2-hydroxypropionyl, formyl,

[0024] C₁₋₄alkoxycarbonyl, C₁₋₁₂alkylcarbonyl, perfluoroC₁₋₄alkyl,C₁₋₄alkylcarbonyl, phenylC₁₋₄alkylcarbon1, substitutedphenylC₁₋₄alkylcarbonyl (where the phenyl substituents are independentlyselected from one or more of, C₁₋₄alkyl, perfluoroC₁₋₄alkyl, C₁₋₄alkoxy,hydroxy, halo amido, nitro amino, C₁₋₄alkylamino, C₁₋₄dialkylamino,carboxy or C₁₋₄alkoxycarbonyl) 1,1-diphenylC₁₋₄alkylcarbonyl,substituted 1,1-diphenylC₁₋₄alkylcarbonyl (where the phenyl substituentsare independently selected from one or more of, C₁₋₄alkyl, perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido, nitro, amino,C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy or C₁₋₄alkoxy-carbonyl),perfluoroC₁₋₄alkylsulfonyl, C₁₋₄alkylsulfonyl, C₁₋₄alkoxysulfonyl,phenylsulfonyl, substituted phenylsulfonyl (where the phenylsubstituents are independently selected from one or more of, C-1alkyl,perfluoro C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy orC₁₋₄alkoxycarbonyl), 10-camphorsulfonyl, phenylC₁₋₄alkylsulfonyl,substituted phenylC₁₋₄alkylsulfonyl, perfluroC₁₋₄alkylsulfinyl,C-14alkylsulfinyl, phenylsulfinyl, substituted phenylsulfinyl (where thephenyl substituents are independently selected from one or more of,C₁₋₄alkyl, perfluoro C₁₋₄alkyl, C₁₋₄ alkoxy, hydroxy, halo, amido,nitro, amino, C₁₋₄ alkylamino, C₁₋₄dialkylamino, carboxy or C₁₋₄alkoxycarbonyl), 1-naphthylsulfonyl, 2-naphthylsulfonyl, substitutednaphthylsulfonyl (where the naphthyl substituents are independentlyselected from one or more of, C₁₋₄alkyl, perfluroC₁₋₄alkyl, C₁₋₄alkoxy,hydroxy, halo, amido, nitro, amino, C₁₋₄alkylamino, C₁₋₄dialkylamino,carboxy or C₁₋₄alkoxycarbonyl),1-naphthysulfinyl, 2-naphthysulfinyl, andsubstituted naphthylsulfinyl (where the naphthyl substituents areindependently selected from one or more of, C₁₋₄alkyl,perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo amido, nitro, amino,C₁₋₄alkylamino, C104dialkylamino, carboxy or C-14alkoxycarbonyl):

[0025] or a poly peptide comprised of two amino acids,

[0026] where the first amino acid is a D or L amino acid, bound via itscarboxy terminus to the nitrogen depicted in Formula I and is selectedfrom the group consisting of glycine, N-C₁₋₈alkyglycine, alanine,2-azetidinecarboxylic acid, proline, thiazolidine-4-carboxylic acid,5.5-dimethylthiazolidine-4-carboxylic acid, oxazolidine-4-carboxylicacid, 1-amino-1-cyclo₃₋₈alkylcarboxylic acid, 3-hydroxypropoline,4-hydroxyproline, 3-(C₁₋₄alkoxy)proline, 4(C₁₋₄alkoxy)proline,3,4-dehydroprline, 2,2-dimethyl-4-thiazolidine carboxylic acid,2.2-dimethyl-4-oxadolidine carboxylic acid, pipecolinic acid, valine,methionine, cysteine, asparagine, serine, threonine, leucine,tert-leucine, isoleucine, phenylalanine, 1-naphthalanine,2-naphthalanine, 2-thienylalanine, 3-thienylane,[1,2,3,4]-tetrahydroisoquinoline-2-carboxylic acid, asparticacid-4-C₁₋₄alkyl ester and glutamic acid 5-C₁₋₄alkyl ester

[0027] and the second D or L amino acid, is bound to the amino terminusof said first amino acid, and is selected from the group consisting ofphenylalanine, 4-benzolyphenylalanine, 4-carboxyphenylalanine,4-(Carboxy C1-2alkyl)phenylalanine, substituted phenylalanine (where thephenyl substituents are independently selected from one or more ofC₁₋₄alkyl, perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido, nitro,amino, C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy or C₁₋₄alkoxycarbonyl),3-benzothienylalanine, 4-biphenylalanine, homophenylalanine,octahydroindole-2-carboxylic acid, 2-pyridylalanine, 3-pyridylalanine,4-thiazolyalanine, 2-thienylalanie, 3-(3-benzothienyl)alanine,3-thienylalanine, tryptophan, tyrosine, asparagine,3-tri-C₁₋₄alkylsilylalanine, cyclohexylglycine, diphenylglycine,phenylglycine, methionine sulfoxide, methionine sulfone,2,2-dicyclohexylalanine, 2-(1-naphthylalanine), 2-(2-naphthylalanine),phenyl substituted phenylalanine (where the substituents are selectedfrom C₁₋₄alkyl, perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido,nitro, amino, C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy orC₁₋₄alkoxycarbonyl), aspartic acid, aspartic acid-C₁₋₄alkyl,perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido, nitro, amino,C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy or C₁₋₄alkoxycarbony),aspartic acid, aspartic acid-4-C₁₋₄alkyl ester glutamic acid, glutamicacid-5-C₁₋₄ alkyl ester, cycloC₃₋₈-alkylalanine, substitutedcycloC₃₋₈alkylalanine (where the ring substituents are carboxy,C₁₋₄alkyl ester, cycloC₃₋₈-alkylalanine, substitutedcycloC₃₋₈alkylalanine (where the ring substituents are carboxy,C₁₋₄alkylcarboxy, C₁₋₄alkoxycarbonyl or aminocarbonyl),2,2-diphenylalanine and all alpha-C₁₋₅alkyl of all amino acidderivatives thereof,

[0028] where the amino terminus of said second amino acid isunsubstituted or monosubstituted with a member of the group consistingof formyl, C1-12 alkyl, tetrazol-5-ylC1-2alkyl, carboxyC1-8 alkyl,carboalkoxyC₁₋₄alkyl, phenyl C₁₋₄alkyl, substituted phenylC₁₋₄alkyl(where the phenyl substituents or independently selected from one ormore of, C₁₋₄alkyl, perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo,amido, nitro, amino, C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy orC₁₋₄alkoxycarbonyl), 1,1-diphenylC₁₋₄alkyl, C1-6alkoxycarbonyl,phenylC1-6alkoxycarbonyl, C1-2alkylcarbonyl,perfluoroC₁₋₄alkylCo-4alkylcarbonyl, phenyC₁₋₄alkylcarbonyl, substitutedphenyC₁₋₄alkylcarbonyl(where the phenyl substituents are independentlyselected from one or more of C₁₋₄alkyl, perfluoro C₁₋₄akyl, C₁₋₄ alkoxy,hydroxy, halo, amido, nitro, amino, C₁₋₄alkylamino, C₁₋₄dialkylamino,carboxy or C₁₋₄alkoxycarbonyl), 1,1-diphenylC₁₋₄alkyl,perfluoroC₁₋₄alkyl, C₁₋₄alkoxycarbonyl), 10-camphorsulfonyl,phenylC₁₋₄alkylsulfonyl, substituted phenylC₁₋₄sulfonyl,C₁₋₄alkysulfmyl, perfluoro C₁₋₄alkylsulfinyl, phenylsulfinyl,substituted phenylsulfinyl (where the phenyl substituents areindependently selected from one or more of, C₁₋₄alkyl,perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido, nitro, amino,C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy or C₁₋₄alkoxycarbonyl),phenyC₁₋₄alkylsulfinyl, substituted phenylC₁₋₄alkylsulfinyl1-naphthylsulfonyl, 2-naphthylsulfonyl, substituted naphthylsulfonyl(where the naphthyl substituent is selected from C₁₋₄alkyl,perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo amido, nitro, amino,C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy or C₁₋₄alkoxycarbonyl),1-naphthyl-sulfonyl, 2-naphthylsulfinyl and substitutednaphthyl-sulfonyl (where the naphthyl substituent is selected fromC₁₋₄alkyl, perfluoroC₁₋₄alkyl, C₁₋₄alkoxy, hydroxy, halo, amido, nitro,amino, C₁₋₄alkylamino, C-14dialkylamino, carboxy or C₁₋₄alkoxycarbonyl);R₁ is selected from the group consisting of hydrogen and alkyl; R₂ isselected from the group consisting of aminoC₂₋₅-alkyl,guanidinoC₂₋₅alkyl, C₁₋₄alkylguanidinoC₂₋₅alkyl,diC₁₋₄alkylguanidinooC₂₋₅alkyl, amidinoC₂₋₅alkyl,C₁₋₄alkyl-amidinoC₂₋₅alkyl, diC₁₋₄alkyl-amidinoC₂₋₅alkyl,C₁₋₃alkoxyC₂₋₅alkyl, phenyl, substituted phenyl (where the substituentsare independently selected from one or more of amino, amidino,guanidino, C₁₋₄alkylamino, C₁₋₄dialkylamino, halogen, perfluoroC₁₋₄alkyl, C₁₋₄akyl, C₁₋₃ alkoxy or nitro), benzyl, phenyl substitutedbenzyl (where the substituents are independently selected from one ormore of, amino, amidino, guanidino, C₁₋₄alkylamino, C₁₋₄dialkyl-amino,halogen, perfluoro C₁₋₄alkyl, C1-04alkyl, C₁₋₃alkoxy or nitro),hydroxyC₂₋₅alkyl, C′₁₋₅aylaminoC₂₋₅alkyl, C₁₋₅dialkylaminoC₂₋₅alkyl,4-aminocyclohexylC₀₋₂alkyl and C₁₋₅alkyl;

[0029] p is 0 or 1;

[0030] where n is 0-3, R₃ is H or C1-5alkyl and the carbonyl moiety of Bis bound to E;

[0031] E is a heterocycle selected from the group consisting ofoxazolin-2-yl, oxazol-2-yl, thiazol-2-yl, thiazol-5-yl, thiazol-4-yl,thiazolin-2-yl, imidazol-2-yl, 4-oxo-2-quinoxalin-2yl, 2-pyridyl,3-pyridyl, benzo[b}thiophen-2-yl, triazol-4-yl triazol-6-yl,pyrazol-2-yl, 4,5,6,7-tetrahydrobenzothiazol-2yl,naphtho[2,1-d]thiazol-2-yl, naphtho[1-2-d]thiazol-2-yl quinoxalin-2-yl,isoquinolin-1-yl, isoquinolin-3-yl, benzo [b]furan-2-yl, [pyrazin-2-yl,quinazolin-2-yl, isothiazol-5-yl, isothiazol-3-yl, purin-8yl and asubstituted heterocycle where the substituents are selected from C₁₋₄from C-14alky, perfluoro C₁₋₄alkyl,C₁₋₄alkoxy, hydroxy, halo, amido,nitro, amino, C₁₋₄alkylamino, C₁₋₄dialkylamino, carboxy,C₁₋₄alkoxycarbonyl, hydroxy or phenylC₁₋₄ alkylaminocarbonyl;

[0032] or pharmaceutically acceptable salts thereof.

[0033] More particularly, in our opinion, some of the compounds of theforegoing formula containing a d-phenylalanine-proline-arginine motifshould be effective in inhibiting the PAR-2 pathway and causingdepigmentation. One particularly preferred compound which acts as athrombin and trypsin inhibitor and is active in depigmenting mammalianskin is(S)-N-Methyl-D-phenylalanine-N-[4-[(aminoiminomethyl)amino]-1-(2-benzothiazoycarbonyl)but]-L-prolinamnide(Chemical Abstracts name) (hereinafter referred to as “Compound I”). Wesuggest that other compounds which are analogs or function similarly toCompound I and are set forth in U.S. Pat. No. 5,523,308 may be active inthe methods and compositions of this invention.

[0034] Other compounds that inhibit trypsin, such as serine proteaseinhibitors, and in particular, soybean trypsin inhibitor (STI) will alsobe useful in methods of this invention. Soybean, limabean and blackbeanextracts, and other natural products made from these beans, such as, butnot limited to, bean milk, bean paste, miso and the like, also serve toreduce pigmentation by this mechanism.

[0035] Additional sources of serine protease inhibitors may be extractedfrom the species belonging to the following plant families: Solanaceae(e.g., potato, tomato, tomatilla, and the like); Gramineae (e.g., rice,buckwheat, sorghum, wheat, barley, oats and the like); Cucurbitaceae(e.g., cucumbers, squash, gourd, luffa and the like); and, preferably,Leguminosae (e.g., beans, peas, lentils, peanuts, and the like).

[0036] While not willing to be bound by the following theory, wetheorize that the compounds capable of affecting the pigmentation of theskin do so by interacting directly or indirectly with the keratinocytePAR-2 or with its activating protease, and thereby affect melanogenesis,directly or indirectly. Possibly, the compounds of this inventioninduce, in the case of increased pigmentation or reduce, in the case ofdecreased pigmentation, the signal to transport melanosomes bymelanocytes, or to receive melanosomes by keratinocytes in the skin.

[0037] Recently we have identified that the Bowman-Birk Inhibitor(“BBI”), a different group of legume-derived proteins, are alsodepigmenting agents.

[0038] While STI is a 21 KD protein with primarily trypsin inhibitoryactivity, the soybean-derived BBI is a smaller, 8 KD protein, whichinhibits chymotrypsin and trypsin. Unlike STI, BBI does not have aKunitz-type domain, suggesting different interactions with serineproteases. BBI is known for its ability to prevent carcinogenesis innumerous in vivo and in vitro models. In some animal carcinogenesismodels BBI was found to have strong anti-inflammatory effects. BBI ismore resistant than STI to heat-denaturation. For a review on BBI seeKennedy A R, Chemopreventive agents: protease inhibitors, Pharmacol Ther78: 3, 167-209, June, 1998.

[0039] The compounds which are active in the compositions and methods ofthis invention may be delivered topically by any means known to those ofskill in the art. If the delivery parameters of the topically activepharmaceutical or cosmetic agent so require, the topically activecomposition of this invention may preferably be further composed of apharmaceutically or cosmetically acceptable vehicle capable offunctioning as a delivery system to enable the penetration of thetopically active agent into the skin.

[0040] One acceptable vehicle for topical delivery of some of thecompositions of this invention, particularly proteins such as trypsinand STI, may contain liposomes. The liposomes are more preferablynon-ionic and contain a) glycerol dilaurate (preferably in an amount ofbetween about 5% and about 70% by weight); b) compounds having thesteroid backbone found in cholesterol (preferably in an amount ofbetween about 5% and about 45% by weight); and c) one or more fatty acidethers having from about 12 to about 18 carbon atoms (preferably in anamount of between about 5% and about 70% by weight collectively),wherein the constituent compounds of the liposomes are preferably in aratio of about 37.5:12.5:33.3:16.7. Liposomes comprised of glyceroldilaurate/cholesterol/polyoxyethylene-10-stearylether/polyoxyethylene-9-lauryl ether (GDL liposomes) are most preferred.Preferably the liposomes are present in an amount, based upon the totalvolume of the composition, of from about 10 mg/mL to about 100 mg/mL,and more preferably from about 20 mg/mL to about 50 mg/mL. A ratio ofabout 37.5:12.5:33.3:16.7 is most preferred. Suitable liposomes maypreferably be prepared in accordance with the protocol set forth inExample 1, though other methods commonly used in the art are alsoacceptable.

[0041] The above described composition may be prepared by combining thedesired components in a suitable container and mixing them under ambientconditions many conventional high shear mixing means well known in theart for non-ionic liposomes preparations, such as those disclosed inNiemiec et al., “Influence of Nonionic Liposomal Composition On TopicalDelivery of Peptide Drugs Into Pilosebacious Units: An In Vivo StudyUsing the Hamster Ear Model,” 12 Pharm. Res. 1184-88 (1995) (“Niemiec”),which is incorporated by reference herein in its entirety. We have foundthat the presence of these liposomes in the compositions of thisinvention may enhance the depigmenting capabilities of some of thecompositions of this invention.

[0042] Other preferable formulations may contain, for example, soybeanmilk or other liquid formulations derived directly from legumes or othersuitable plant. For example, such a formulation may contain a largeproportion of soybean milk, an emulsifier that maintains the physicalstability of the soybean milk, and, optionally a chelating agent,preservatives, emollients, humectants and/or thickeners or gellingagents.

[0043] Oil-in-water emulsions, water-in-oil emulsions, solvent-basedformulations and aqueous gels known to those of skill in the art mayalso be utilized as vehicles for the delivery of the compositions ofthis invention.

[0044] The source of active compound to be formulated will generallydepend upon the particular form of the compound. Small organic moleculesand peptidyl fragments can be chemically synthesized and provided in apure form suitable for pharmaceutical/cosmetic usage.

[0045] Products of natural extracts can be purified according totechniques known in the art. Recombinant sources of compounds are alsoavailable to those of ordinary skill in the art.

[0046] In alternative embodiments, the topically active pharmaceuticalor cosmetic composition may be optionally combined with otheringredients such as moisturizers, cosmetic adjuvants, anti-oxidants,bleaching agents, tyrosinase inhibitors and other known depigmentationagents, surfactants, foaming agents, conditioners, humectants,fragrances, viscosifiers, buffering agents, preservatives, sunscreensand the like. The compositions of this invention may also contain activeamounts of retinoids (i.e., compounds that bind to any members of thefamily of retinoid receptors), including, for example, tretinoin,retinol, esters of tretinoin and/or retinol and the like.

[0047] The topically active pharmaceutical or cosmetic compositionshould be applied in an amount effective to affect changes in thepigmentation of mammalian skin. As used herein “amount effective” shallmean an amount sufficient to cover the region of skin surface where achange in pigmentation is desired. Preferably, the composition isliberally applied to the skin surface such that, based upon a square cmof skin surface, from about 2 μl/cm² to about 200 μl/cm² of topicallyactive agent is present when a change in pigmentation is desired. Whenusing a thrombin and trypsin inhibitor such as Compound I or itsanalogs, whether synthetically- or naturally-derived in a formulation,such an active compound should be present in the amount of from about0.0001% to about 15% by weight/volume of the composition. Morepreferably, it should be present in an amount from about 0.0005% toabout 5% of the composition; most preferably, it should be present in anamount of from about 0.001 to about 1% of the composition. Of course,these ranges are suggested for the foregoing components. The lower setof ranges is intended to be efficacious for PAR-2 pathwayagonists/antagonists and/or inhibitors having high therapeutic indicesand which do not require significantly larger concentrations or doses tobe effective in the methods of this invention. Such compounds may besynthetically- or naturally-derived.

[0048] Liquid derivatives and natural extracts made directly from plantsor botanical sources may be employed in the compositions of thisinvention in a concentration (w/v) from about 1 to about 99%. Fractionsof natural extracts and naturally-derived protease inhibitors such asSTI may have a different preferred range, from about 0.01% to about 20%and, more preferably, from about 1% to about 10% of the composition. Ofcourse, mixtures of the active agents of this invention may be combinedand used together in the same formulation, or in serial applications ofdifferent formulations.

[0049] We have unexpectedly found that when topically active agents,such as PAR-2 agonists and/or inhibitors and trypsin and/or thrombinand/or tryptase and/or their inhibitors, are topically applied to ananimal's skin, a significant change in pigmentation was achieved.Preferably, depigmenting agents (as well as other pigmentation-affectingagents of this invention) are applied to the skin of a mammal at arelatively high concentration and dose (from about 0.005% to about 1%for compounds having high therapeutic indices such as Compound I andrelated compounds; from about 20% to about 99% for liquid derivativesand extracts of botanical materials; and from about 1% to about 20% forfractions of natural extracts and naturally-derived protease inhibitorssuch as STI or mixtures thereof) between one and two times daily for aperiod of time until the skin evidences a change in pigmentation. Thismay be for from about four to about ten weeks or more. Thereafter, oncethe change in pigmentation has been achieved, a lower concentration anddose (from about 0.00001% to about 0.005% for compounds having hightherapeutic indices such as Compound I and related compounds; from about10% to about 90% for liquid derivatives and extracts of botanicalmaterials; and from about 0.01% to about 5% for fractions of naturalextracts and naturally-derived protease inhibitors such as STI ormixtures thereof), of active ingredient may be applied on a lessfrequent time schedule, e.g., about once per day to about twice perweek. The effects of the active agents of this invention are reversible,therefore, in order to maintain these effects, continuous application oradministration should be performed. The invention illustrativelydisclosed herein suitably may be practiced in the absence of anycomponent, ingredient, or step which is not specifically disclosedherein.

[0050] The invention illustratively disclosed herein suitably may bepracticed in the absence of any component, ingredient, or step which isnot specifically disclosed herein. Several examples are set forth belowto further illustrate the nature of the invention and the manner ofcarrying it out, but do not serve to limit the scope of the methods andcompositions of this invention.

EXAMPLE 1

[0051] BBI Affects Pigmentation

[0052] In order to study the possible roles of BBI in pigmentation, anin N epidermal equivalent system containing melanocytes was used. Theepidermal equivalent system used in this study is the MelanoDerm mel-300system, available commercially from MatTek Co. of Ashland, Mass. Thissystem contains human normal melanocytes, together with normal,human-derived epidermal keratinocytes, derived from African-Americanforeskin. These cells have been cultured to form a multi-layered, highlydifferentiated model of the human epidermis. In the following examples,equivalents were treated with BBI (0.1%) for three days and samples wereharvested on the fourth day after beginning of treatment. The harvestedequivalents were first compared for their color without staining,following by histological examination with Fontana-Mason (F&M) staining,a stain known to those of skill in the art. F&M staining is a silverstaining technique that clearly and cleanly marks melanins which havehigh silver nitrate reducing activity. Images of the stained sectionswere also captured for image analysis. At least three sections perequivalent, three equivalents per experiment were processed. EmpireImages database 1.1 was used on a Gateway 2000 P5-100 computer (MediaCybernetics, Silver Springs, Md.) for capturing images. Image Pro Plusversion 3.0 was used for image analysis. Parameters measured were thesurface area of silver deposits within melanocytes and the densityluminosity of each pixel. A “pigmentation factor” was defined as thesurface area of silver deposits divided by the total epidermal surfacearea A value of one (100%) was assigned to untreated controls, andvalues of treatment groups were normalized to their relevant controls.

[0053] As shown in FIG. 1, untreated mel-300 equivalents are visiblydark without any staining. BBI treated equivalents were lighter thanthese controls, demonstrating the ability of BBI to visually reducepigmentation. FIG. 2 shows the histological sections of theseequivalents, following F&M staining. In this Figure, black areasrepresent melanin deposits within both melanocytes and keratinocytes. Asshown in FIG. 2, BBI treatment results in reduced melanin depositionboth in the melanocytes and in the keratinocytes of the treatedequivalents. Image analysis revealed that BBI treated equivalents haveonly 50.6% melanin deposits relative to controls.

EXAMPLE 2

[0054] The depigmenting effect of BBI is dose-responsive.

[0055] Epidermal equivalents containing melanocytes as described inexample 1 were treated with increasing concentrations of BBI, from0.001% to 0.1%. Following the same experimental procedure described inexample 1, the depigmenting effect of BBI was found to bedose-dependent. FIG. 3 shows F&M stained sections of the treatedequivalents, demonstrating the dose-response and the depigmenting effectof as low as 0.001% BBI. Computerized image analysis, shown in FIG. 4,quantifies this effect and further demonstrates its dose-responsivenature.

EXAMPLE 3

[0056] In vivo demonstration of the depigmenting effect of BBI

[0057] A dark skin Yucatan microswine was treated with BBI, or STI, 1%,in PBS, with 20/mg/ml liposomes. Non-ionic liposomes preparations, suchas those disclosed in Nieriec et al., “Influence of Nonionic LiposomalComposition On Topical Delivery of Peptide Drugs Into PilosebaciousUnits: An In Vivo Study Using the Hamster Ear Model,” 12 Pham. Res.1184-88 (1995) (“Niemiec”), which is incorporated by reference herein inits entirety, are well known in the art, and are described in JBP-430.We have found that the presence of these liposomes in the compositionsof this invention may enhance the depigmenting capabilities of some ofthe compositions of this invention. GDL liposomes were prepared as setforth in Niemiec, et al., above, with the exception of the followingchanges: the non-ionic liposomal formulation contained glyceroldilaurate (Emulsynt GDL, ISP Van Dyk)/cholesterol(Croda)/polyoxyethylene-10-stearyl ether (Brij76,ICI)/polyoxyethylene-9-laurylether, as at ratio of 37.5:12.5:33.3:16.7.Hepes buffer, 0.05M, pH 7.4 (Gibco-BRL of Gaithersburg, Md.) was used asthe aqueous phase in the preparation of the liposomes.

[0058] The BBL STI and liposome vehicle preparations were applied eachonto two sites of the swine's flank, twice daily, five days per week,for eight weeks. After eight weeks of treatment, the application ofeither BBI or STI resulted in a visible lightening effect. Histologicalanalysis of F&M stained skin sections from untreated and treated sitesconfirmed this observation. FIG. 5 shows the F&M stained skin sectionsof the treated swine, demonstrating a dramatic reduction in pigmentdeposition in sites treated with BBI or STI. Computerized imageanalysis, shown in FIG. 6, quantifies this effect and furtherdemonstrates the depigmenting effect of BBI.

What is claimed is:
 1. A method of effecting changes in mammalian skinpigmentation comprising administering to a mammal apigmentation-changing effective amount of a Bowman-Birk Inhibitor or ofa natural extract containing a Bowman-Birk Inhibitor.
 2. A method ofdepigmenting mammalian skin pigmentation comprising administering to amammal a pigmentation-lightening effective amount of a Bowman-BirkInhibitor or of a natural extract containing a Bowman-Birk Inhibitor. 3.A method according to claim 2 wherein said Bowman-Birk Inhibitor isderived from one or more of the botanical families legurrinosae,solanaceae, gramineae and cucurbitaceae.
 4. A method according to claim3 wherein said compound is derived from legumes.
 5. A method accordingto claim 4 wherein said compound is derived from undenatured soybeanextract.
 6. A method according to claim 5 wherein said compound isderived from fractions of undenatured soybean extract.
 7. A compositionfor affecting changes in mammalian skin pigmentation comprising apigmentation-changing effective amount of a Bowman-Birk Inhibitor or ofa natural extract containing a Bowman-Birk Inhibitor.
 8. A compositionfor depigmenting mammalian skin pigmentation comprising administering toa mammal a pigmentation-lightening effective amount of a Bowman-BirkInhibitor or of a natural extract containing a Bowman-Birk Inhibitor. 9.A composition according to claim 8 wherein said Bowman-Birk Inhibitor isderived from one or more of the botanical families leguminosae,solanaceae, gramineae and cucurbitaceae.
 10. A composition according toclaim 9 wherein said compound is derived from legumes.
 11. A compositionaccording to claim 10 wherein said compound is derived from undenaturedsoybean extract.
 12. A composition according to claim 11 wherein saidcompound is derived from fractions of undenatured soybean extract.
 13. Acomposition according to claim 7 wherein said Bownian-Birk Inhibitor ispresent in an amount of from about 0.0001% to about 20% by weight/volumeof said composition.
 14. A composition according to claim 13 whereinsaid compound is present in an amount from about 0.001 to about 15% ofsaid composition.
 15. A composition according to claim 14 wherein saidcompound is present in an amount from about 0.005 to about 1% of saidcomposition.
 16. A method according to claim 1 wherein said compositionis applied at least once daily for at least eight weeks.
 17. A methodaccording to claim 16 wherein said composition is applied at arelatively high dosage for at least about four to about ten weeks andthen applied at a relatively lower dosage on a continuous basis tomaintain skin lightening effect.
 18. A method according to claim 1wherein said composition is administered orally.
 19. A method accordingto claim 1 wherein said composition is administered parenterally.
 20. Acosmetic composition according to claim 7 comprising saidpigmentation-affecting compound and a cosmetically-acceptable vehicle.21. A composition according to claim 20 wherein said composition furthercomprises additional depigmenting agents.
 22. A composition according toclaim 21 wherein said composition further comprises tyrosinaseinhibitors.
 23. A composition according to claim 20 wherein saidcomposition further comprises liposomes.
 24. A composition according toclaim 23 wherein said composition comprises glycerol dilaurate,cholesterol, polyoxyethylene-10-stearyl ether andpolyoxyethylene-9-lauryl ether.
 25. A composition according to claim 20wherein said composition further comprises anti-oxidants.
 26. Acomposition according to claim 20 wherein said composition furthercomprises a sunscreen.
 27. A composition according to claim 20 whereinsaid composition further comprises a compound selected from the groupconsisting of: anti-oxidants, sunscreens, moisturizers, bleachingagents, depigmentation agents, surfactants, foaming agents,conditioners, humectants, fragrances, viscosifiers, buffering agents,preservatives and a mixture thereof.